Psma-targeting imaging agents

ABSTRACT

A PSMA-specific imaging agent comprising a compound according to formula I: 
     
       
         
         
             
             
         
       
     
     are described, wherein S 1  is an organic spacer group having from 5 to 30 carbons, A is an amino acid forming a portion of a negatively charged peptide oligomer, n is from 3 to 6, S 2  is an organic spacer group having from 5 to 15 carbons, and I is an imaging group, and pharmaceutically acceptable salts thereof. The PSMA-specific imaging agents can be used to image PSMA within a tissue region to guide the treatment of diseases such as prostate cancer.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication 61/931,112 filed on Jan. 24, 2014, and entitledBIODISTRIBUTION OF PSMA-TARGETING TRACERS WITH A HIGHLY NEGATIVELYCHARGED LINKER, the entirety of which is incorporated by referenceherein.

GOVERNMENT FUNDING

This invention was made with government support under Army Grant#W81XWH-10-1-0218, awarded by the U.S. Department of Defense. Thegovernment has certain rights in the invention

BACKGROUND

Prostate cancer is the most common malignancy in males and the secondleading cause of death from cancer in men. It is estimated that therewill be 238,590 new cases of prostate cancer in 2013. The majority ofthese patients will undergo definitive treatment. However, about 35% areexpected to have biochemical recurrence within 10 years. This translatesto nearly 70,000 patients a year with PSA recurrence after definitivetherapy. Currently, no commercially available molecular imaging agentcan effectively localize regional prostate metastases in soft tissue.Talab et al., Radiol Clin North Am; 50(6):1015-1041 (2012). Thedevelopment of a sensitive and specific method to non-invasivelylocalize prostate cancer in its early stages within the prostate and inlocal pelvic lymph nodes would profoundly change the workup andmanagement of prostate cancer.

Small molecule inhibitors of prostate specific membrane antigen (PSMA)have shown the potential to be good agents for prostate cancer imaging.PSMA is a type II membrane protein with a very short intracellulardomain connected by a single transmembrane helix to a largeextracellular domain. Israeli et al., Cancer Res; 53(2):227-230 (1993).PSMA was first identified as the molecular target of the 7E11-C5antibody which selectively binds LNCaP cells. In addition to its normalexpression in the central nervous system, urogenital system, and smallbowel, PSMA is over-expressed on prostate cancer cells and tumorneovasculature. A simple, easy to synthesize, and yet potent, urea-basedsmall molecule inhibitor of PSMA was first published in 2001. Kozikowskiet al., J Med Chem; 44(3):298-301 (2001). During the last decade, thesimple di-amino acid urea compounds first made by Kozikowski et al. haveevolved into a myriad of imaging agents for single photon emissiontomography (SPECT) and positron emission tomography (PET).

Small molecule PET and SPECT PSMA tracers that have been tested inanimals and humans have demonstrated a great advancement compared toantibody-based SPECT imaging with ¹¹¹In-capromab pendetide. ¹⁸F-DCFBCwas the first PSMA-targeting PET tracer to be tested in humans. Cho etal., J Nucl Med; 53(12):1883-1891 (2013). In a small five-patient trial,¹⁸F-DCFBC detected lymph node and bone metastases at 2 hr postinjection. In a seven-patient phase 1 study of ¹²³I-MIP-1072 and¹²³I-MIP-1095, the SPECT tracer also demonstrated detection of softtissue and bone metastases, as well as tumors in the prostate bed.Barrett et al., J Nucl Med; 54(3):380-387 (2013). Afshar-Oromieh et al.tested Glu-NH—CO—NHLys(Ahx)-[⁶⁸Ga(HBED-CC)] in 37 patients with prostatecancer and demonstrated a (per patient) lesion detection rate of 60% atPSA<2.2 ng/ml and a detection rate of 100% at PSA>2.2 ng/ml.Afshar-Oromieh et al., Eur J Nucl Med Mol Imaging; 40(4):486-495 (2013).All of these early human trials showed good lesion to backgroundcontrast at a few hours post injection compared to ¹¹¹In-capromabpenditide images, which need to be acquired 4 days post injection.However, ¹⁸F-DCFBC also had elevated liver background and unexpectedblood pool retention. ¹²³I-MIP-1072, ¹²³I-MIP-1095, andGlu-NH—CO—NH-Lys(Ahx)-[⁶⁸Ga(HBEDCC) all showed significant uptake insalivary glands, lacrimal glands, and liver. These organs have nosignificant expression of PSMA. When the tracer molecules are used asdiagnostic agents, the elevated background affects the overallsensitivity of detection. If these agents would be used for therapy,unintended background would increase the overall toxicity of thetreatment.

The non-PSMA related background activity exhibited by the currenttracers may be due to hydrophobic interactions. Small radiolabeled PSMAtracers contain an aromatic group for convenient radiohalogenation. Manyof the imaging agents with bulky NIR fluorophores and radionuclidechelates have long slender hydrophobic linkers that join the fluorophoror metal chelate to the di-amino acid urea moiety. A long linker isnecessary because a 20 Å substrate tunnel connects the surface of PSMAwith its deep ectodomain. Mesters et al., EMBO J.; 25(6):1375-1384(2006). Early design efforts with ^(99m)Tc tracers demonstrated thatthere is a minimal linker length needed for proper binding. Banerjee etal., J. Med Chem; 51 (15):4504-4517 (2008). In the available highresolution structures (such as PDB 3D7G and 3D7H), where space allows,only a few crystallographic water molecules are seen within the tunnel.Given the known structure of PSMA, one would expect low binding affinityfor RBI-1033, a urea-based PSMA targeting compound containing a bulky2-5 Å moiety in the substrate tunnel region. Cramer NucleosidesNucleotides Nucleic Acids; 26(10-12):1471-1477 (2007). The axialdimension of the 2-5 Å moiety greatly exceeds the width of the tunnel.

Surprisingly, RBI1033 exhibits ten times higher affinity toward PSMAthan its “parent” urea ligand. The high affinity of RBI-1033 and itsderivatives to PSMA suggest that a bulky linker is acceptable in thesubstrate tunnel. Wang et al., Nucleosides Nucleotides Nucleic Acids;31(5):432-444 (2012). However, there remains a need for additionalcompounds useful as PSMA imaging agents.

SUMMARY OF THE INVENTION

Through iterative redesign, a series of PSMA inhibitors were designedwith highly negatively charged linkers that connect to urea inhibitorsand bulky radionuclide chelates. In vivo imaging and biodistributionstudies were then performed with the radiolabeled tracers. The tracersderived from the iterative redesign have affinities for PSMA comparableto the “parent” urea ligand Cys-C(O)-Glu. Using a fluorine-18 labeledPSMA targeting tracer, it was found that these highly negatively chargedmolecules exhibit rapid renal excretion with minimal non-specificbinding. The biodistribution data at 2 hr showed 4.6% ID/g PC3-PIP tumoruptake with spleen, liver, bone, and blood background levels of 0.1%,0.17%, 0.1%, and 0.04%, respectively.

Placement of multiple negative charges in the linker region of PSMAtracers significantly reduced the non-specific background bindingwithout significant reduction of binding affinity. This increasedtumor/background contrast in positron emission tomography promises toprovide more sensitive tumor detection while decreasing the overallradiation exposure to patients.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a scheme showing the chemical structures ofRBI-1033(Top), ZJ-MCC-Ahx-EEEG (compound 1), ZJ-MCC-Ahx-YYYG (compound2), E′EAmc-Ahx-dEdEdEGYGGGC in the presumed structure of the technetiumbound form [^(99m)Tc]3, E′EAmc-Ahx-EEEYK(Bn-NOTA) (compound 4) and thepresumed structure of E′E-Ahx-EEEYK(Bn-NOTA) with a boundaluminum-fluoride complex ([Al¹⁸F]5).

FIG. 2 shows a MicroSPECT/CT image of an NSG mouse with PC3-PIP(PSMA+)and PC3-flu(PSMA−) xenographs. Images were acquired 4 hours after tailvein injection of radiolabled [^(99m)Tc]3. (Left) an axial slice ofSPECT/CT through the axillary region of the mouse demonstrating a PSMA+tumor on the right side and a PSMA− tumor on the left side. (Right) amaximum intensity projection (MIP) image of the mouse. Expected normalphysiologic accumulation is noted in the kidneys, bowel and bladder inaddition to the PSMA+ tumor. The images demonstrate near backgroundtracer uptake in the PSMA negative tumors, liver, lung, heart andspleen.

FIG. 3 provides a PET/CT image of an NSG mouse with two PC3-PIP(PSMA+)xenographs. Images were acquired 1 hours after tail vein injection of[Al¹⁸F]5. The middle panel is maximum intensity projection image (MIP);the left panel is MIP overlayed with CT. The actual tumors are shown inthe right panel. The smaller tumor has a maximum dimension of 3 mm.Expected normal physiologic accumulation is noted in the kidneys andbladder. The images demonstrate minimal background tracer uptake in theliver, lung, heart and spleen.

FIG. 4 provides a general schematic representation of the elongated PSMAbinding tracers. For ZJ-MCCdEdEdEGK(IRDye800cw)G, R1, R2, and R3 areD-glutamic acid residues; IRDye800cw is attached at the R4 position.

FIG. 5 shows preoperative white light (A) and near-infrared (NIR)fluorescence (B) imaging with the Maestro in vivo imaging system. Notethe strong fluorescence signal from the tracer in the right flank (PSMApositive tumor), bladder, and urethra, and the background fluorescencesignal from the kidneys. Arrows indicate bulging of the tumor in theskin.

FIG. 6 provides intraoperative images of prostate tumors excised frommice using the da Vinci Si system with a fluorescence detectingendoscope. (A) Fluorescent staining of a PSMA-positive tumor implantusing a PSMA binding, fluorescently tagged compound (arrows). (B)Nonstaining of a PSMA negative (control) tumor implant.

DETAILED DESCRIPTION OF THE INVENTION

Through iterative re-design of RBI-1033, the inventors found that thelinker region is amenable to further engineering and one can constructPSMA tracers with relatively bulky negatively charged linker regions.The inventors further demonstrated that such a negatively charged agentexhibits an improved biodistribution profile with a lower overallbackground compared to existing agents in the literature.

DEFINITIONS

The terminology as set forth herein is for description of theembodiments only and should not be construed as limiting of theinvention as a whole. Unless otherwise specified, “a,” “an,” “the,” and“at least one” are used interchangeably. Furthermore, as used in thedescription of the invention and the appended claims, the singular forms“a”, “an”, and “the” are inclusive of their plural forms, unlesscontraindicated by the context surrounding such.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

“Image” or “imaging” refers to a procedure that produces a picture of anarea of the body, for example, organs, bones, tissues, or blood.

“Pharmaceutically acceptable” as used herein means that the compound orcomposition is suitable for administration to a subject for the methodsdescribed herein, without unduly deleterious side effects.

As used herein, “a detectably effective amount” of the imaging agent ofthe invention is defined as an amount sufficient to yield an acceptableimage using equipment which is available for clinical use. A detectablyeffective amount of the imaging agent of the invention may beadministered in more than one injection. The detectably effective amountof the imaging agent of the invention can vary according to factors suchas the degree of susceptibility of the individual, the age, sex, andweight of the individual, idiosyncratic responses of the individual, andthe dosimetry. Detectably effective amounts of the imaging agent of theinvention can also vary according to instrument and film-relatedfactors. Optimization of such factors is well within the level of skillin the art. The amount of imaging agent used for diagnostic purposes andthe duration of the imaging study will depend upon the radionuclide usedto label the agent, the body mass of the patient, the nature andseverity of the condition being treated, the nature of therapeutictreatments which the patient has undergone, and on the idiosyncraticresponses of the patient. Ultimately, the attending physician willdecide the amount of imaging agent to administer to each individualpatient and the duration of the imaging study.

The terms “comprising” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

As used herein, the term “organic group” is used for the purpose of thisinvention to mean a hydrocarbon group that is classified as an aliphaticgroup, cyclic group, or combination of aliphatic and cyclic groups(e.g., alkaryl and aralkyl groups). In the context of the presentinvention, suitable organic groups for PSMA-specific imaging agents arethose that do not interfere with the compounds activity. In the contextof the present invention, the term “aliphatic group” means a saturatedor unsaturated linear or branched hydrocarbon group. This term is usedto encompass alkyl, alkenyl, and alkynyl groups, for example.

As used herein, the terms “alkyl”, “alkenyl”, and the prefix “alk-” areinclusive of straight chain groups and branched chain groups and cyclicgroups, e.g., cycloalkyl and cycloalkenyl. Unless otherwise specified,these groups contain from 1 to 20 carbon atoms, with alkenyl groupscontaining from 2 to 20 carbon atoms. In some embodiments, these groupshave a total of at most 10 carbon atoms, at most 8 carbon atoms, at most6 carbon atoms, or at most 4 carbon atoms. Lower alkyl groups are thoseincluding at most 6 carbon atoms. Examples of alkyl groups includehaloalkyl groups and hydroxyalkyl groups.

Unless otherwise specified, “alkylene” and “alkenylene” are the divalentforms of the “alkyl” and “alkenyl” groups defined above. The terms,“alkylenyl” and “alkenylenyl” are used when “alkylene” and “alkenylene”,respectively, are substituted. For example, an arylalkylenyl groupcomprises an alkylene moiety to which an aryl group is attached.

Cycloalkyl groups are cyclic alkyl groups containing 3, 4, 5, 6, 7 or 8ring carbon atoms like cyclopropyl, cyclobutyl, cyclopentyl, cyclohexylor cyclooctyl, which can also be substituted and/or contain 1 or 2double bounds (unsaturated cycloalkyl groups) like, for example,cyclopentenyl or cyclohexenyl can be bonded via any carbon atom.

The term “aryl” as used herein includes carbocyclic aromatic rings orring systems. Examples of aryl groups include phenyl, naphthyl,biphenyl, anthracenyl, phenanthracenyl, fluorenyl and indenyl. Arylgroups may be substituted or unsubstituted.

Unless otherwise indicated, the term “heteroatom” refers to the atoms O,S, or N.

When a group is present more than once in any formula or schemedescribed herein, each group (or substituent) is independently selected,whether explicitly stated or not. For example, for the formula —C(O)—NR₂each R group is independently selected.

As a means of simplifying the discussion and the recitation of certainterminology used throughout this application, the terms “group” and“moiety” are used to differentiate between chemical species that allowfor substitution or that may be substituted and those that do not soallow for substitution or may not be so substituted. Thus, when the term“group” is used to describe a chemical substituent, the describedchemical material includes the unsubstituted group and that group withnonperoxidic O, N, S, Si, or F atoms, for example, in the chain as wellas carbonyl groups or other conventional substituents. Where the term“moiety” is used to describe a chemical compound or substituent, only anunsubstituted chemical material is intended to be included. For example,the phrase “alkyl group” is intended to include not only pure open chainsaturated hydrocarbon alkyl substituents, such as methyl, ethyl, propyl,tert-butyl, and the like, but also alkyl substituents bearing furthersubstituents known in the art, such as hydroxy, alkoxy, alkylsulfonyl,halogen atoms, cyano, nitro, amino, carboxyl, etc. Thus, “alkyl group”includes ether groups, haloalkyls, nitroalkyls, carboxyalkyls,hydroxyalkyls, sulfoalkyls, etc. On the other hand, the phrase “alkylmoiety” is limited to the inclusion of only pure open chain saturatedhydrocarbon alkyl substituents, such as methyl, ethyl, propyl,tert-butyl, and the like.

The term “amino acid” as used herein is understood to mean an organiccompound containing both a basic amino group and an acidic carboxylgroup. Included within this term are natural amino acids (e.g., L-aminoacids), modified and unusual amino acids (e.g., D-amino acids), as wellas amino acids which are known to occur biologically in free or combinedform but usually do not occur in proteins. Natural amino acids include,but are not limited to, alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, serine, threonine, tyrosine,tyrosine, tryptophan, proline, and valine. Other amino acids include,but not limited to, arginosuccinic acid, citrulline, cysteine sulfinicacid, 3,4-dihydroxyphenylalanine, homocysteine, homoserine, ornithine,carnitine, selenocysteine, selenomethionine, 3-monoiodotyrosine,3,5-diiodotryosine, 3,5,5′-triiodothyronine, and3,3′,5,5′-tetraiodothyronine.

Compounds described herein can exist or be converted to apharmaceutically acceptable salt. The salts can be prepared by treatingthe free acid with an appropriate amount of a chemically orpharmaceutically acceptable base.

Representative chemically or pharmaceutically acceptable bases areammonium hydroxide, sodium hydroxide, potassium hydroxide, lithiumhydroxide, calcium hydroxide, magnesium hydroxide, ferrous hydroxide,zinc hydroxide, copper hydroxide, aluminum hydroxide, ferric hydroxide,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, lysine, arginine, histidine, and the like. In oneaspect, the reaction is conducted in water, alone or in combination withan inert, water-miscible organic solvent, at a temperature of from about0° C. to about 100° C. (e.g. at room temperature). The molar ratio ofthe compound to base used is chosen to provide the ratio desired forparticular salts. For preparing, for example, the ammonium salts of thefree acid starting material, the starting material can be treated withapproximately one equivalent of base to yield a salt.

Certain compounds described herein may exist in particular geometric orstereoisomeric forms. The present invention contemplates all suchcompounds, including cis- and trans-isomers, R- and S-enantiomers,diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof,and other mixtures thereof, as falling within the scope of theinvention. Additional asymmetric carbon atoms may be present in asubstituent such as an alkyl group. All such isomers, as well asmixtures thereof, are intended to be included in this invention.

Prostate specific membrane antigen (PSMA) is a type II membrane proteinwith a very short intracellular domain connected by a singletransmembrane helix to a large extracellular domain. PSMA isoverexpressed on most solid tumor neovasculature, as well as in prostatecancer, and is therefore a useful target for imaging agents. Chang etal., Cancer Res. 59, 3192-3198 (1999). For further information regardingthe prostate specific membrane antigen, see US Patent Publication No.2007/0148662, the disclosure of which is incorporated herein byreference.

PSMA-Specific Imaging Agents

In one aspect, the present invention provides PSMA-specific imagingagents, which are compounds according to formula I:

wherein S¹ is an organic spacer group having from 5 to 30 carbons, A isa negatively charged amino acid forming a portion of a peptide oligomer,n is from 3 to 6, S² is an organic spacer group having from 5 to 15carbons, and I is an imaging group, and pharmaceutically acceptablesalts thereof.

PSMA-specific, as used herein, refers to the fact that imaging agentsspecifically bind to PSMA rather than other biomaterial. As used herein,the term “specifically binding” refers to the interaction of the imagingagent with a second chemical species, wherein the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,the imaging agent recognizes and binds to a specific protein structureof PSMA rather than to proteins generally.

The PSMA-specific imaging agents include three main regions: the PSMAbinding region, a spacer region including the organic spacer groups andthe negatively charged peptide oligomer, and the imaging group region.The PSMA binding region includes a urea ligand based on Cys-C(O)-Glu,and plays an important role in the specific binding of the imagingagent.

The spacer region serves to separate the imaging group and the PSMAbinding region, but also plays a role in the specific binding of thePSMA-specific imaging agent. In addition, as described herein, thenegatively charged peptide oligomer within the spacer region improvesthe biodistribution profile of the PSMA-specific imaging agent byreducing background binding. The negatively charged peptide oligomer isnegatively charged as a result of including one or more negativelycharged amino acids. Examples of negatively charged amino acids includeglutamic acid and aspartic acid. A preferred negatively charged aminoacid is glutamic acid, with D-glutamic acid being used in someembodiments. One or more of the amino acids making up the negativelycharged peptide oligomer can be negatively charged amino acids. In apreferred embodiment, three of the amino acids are negatively charged.The amino acids are linked through peptide bonds to form a relativelyshort negatively charged peptide oligomer having a length of 3 to 6amino acids. However, lengths of 3, 4, 5, 6, 3-5, 3-4, 4-5, 4-6, and 5-6amino acids are also within the scope of the present invention. Notethat further amino acids can also be present in the adjacent spacergroups.

Adjacent to the negatively charged amino acid peptide oligomer are twoorganic spacer groups, designated S¹ and S² in formula 1. The organicspacer groups S¹ and S² connect the negatively charged peptide oligomerto the PSMA binding region and the imaging group, respectively. Theorganic spacer groups can include from 5 to 30 carbons, from 5 to 15carbons, or from 10 to 15 carbons. The organic spacer groups areprimarily long alkyl chains, but in some embodiments the organic spacergroup can include an aryl group such as a phenyl ring. Typically, thebulk of the organic spacer groups are formed through the reaction ofamino acids with one another. Accordingly, in some embodiments, theorganic spacers include one or more amino acids. The organic spacergroups can include both natural and non-natural amino acids,oligopeptides, for example, linear or cyclic oligopeptides, and nucleicacids. The organic spacer group can be a peptide or peptide moiety.

A common organic spacer for S¹ is an organic spacer group having thestructure:

while a common organic spacer for S² includes a tyrosine-lysinedipeptide.

The PSMA-specific imaging agent also includes an imaging group, which isa structure that allows the imaging agent to be detected using anappropriate imaging device. Examples of imaging agents include nearinfrared imaging agents, positron emission tomography imaging agents,single-photon emission tomography agents, fluorescent compounds,radioactive isotopes, and MRI contrast agents. The detectable imaginggroup can be any material having a detectable physical or chemicalproperty. Such imaging groups have been well-developed and, in general,most any imaging group can be used in the present invention. Thus, animaging group is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. The choice of imaging group depends on sensitivityrequired, ease of conjugation with the compound, stability requirements,available instrumentation, and disposal provisions. A comprehensivereview of imaging agents and their imaging groups can be found in theMolecular Imaging and Contrast Agents Database (MICAD), developed by theNational Center for Biotechnology Information, which is incorporatedherein by reference.

Various fluorochromes are commercially available and can be used as nearinfrared imaging groups for the imaging agents of the invention.Exemplary fluorochromes include, for example, Cy5.5, Cy5 and Cy7 (GEHealthcare); AlexaFlour660, AlexaFlour680, AlexaFluor750, andAlexaFluor790 (Invitrogen); VivoTag680, VivoTag-5680, and VivoTag-S750(PerkinElmer); Dy677, Dy682, Dy752 and Dy780 (Dyomics); DyLight547,DyLight647 (Pierce); HiLyte Fluor 647, HiLyte Fluor 680, and HiLyteFluor 750 (AnaSpec); IRDye 800CW, IRDye 800RS, and IRDye 700DX (Li-Cor);and ADS780WS, ADS830WS, and ADS832WS (American Dye Source) and KodakX-SIGHT 650, Kodak X-SIGHT 691, Kodak X-SIGHT 751 (Carestream Health).An example of a PSMA-specific imaging agent including a near-infraredimaging group is ZJ-MCC-dEdEdEGK(IRDye800cw)G.

In some embodiments, the imaging group comprises a radioisotope.Specific exemplary radioisotopes include ¹⁸F, ¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁶I,¹³¹I, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁸⁰Br, ^(80m)Br, ⁸²Br, ⁸³Br, ⁶⁸Ga and ²¹¹At.Radioisotope containing compounds of any embodiment of the presentinvention can be prepared with sufficient radiolabel to be used inimaging applications. In other words, the compounds can be prepared withradioisotope concentrations greater than natural abundance, when aparticular radioisotope occurs naturally.

Radiolabeled compounds may be used for diagnostic, imaging, ortherapeutic purposes. For example, some compounds, e.g. those labeledwith ¹²⁵I and ¹²³I, are designed for SPECT imaging, while somecompounds, e.g. those labeled with ¹⁸F, ⁶⁸Ga, and ¹²⁴I, are designed forPET imaging, and some radioisotopically labeled compounds may be usedtherapeutically. In general, the suitability of a particularradioisotope for a particular purpose is well understood in the art.

In some embodiments, the imaging group is a positron or single-photonemission tomography imaging group. Examples of imaging agents includingpositron or single-photon emission tomography imaging group includeE′E-Amc-Ahx-dEdEdEGYGGGC-NH₂, E′E-Amc-Ahx-dEdEdEYK(Bn-NOTA)-NH₂, andE′E-Ahx-EEEYK(Bn-NOTA)-NH₂.

In some embodiments, the imaging group is suitable for use as a magneticresonance imaging agent. Disease detection using MRI is often difficultbecause areas of disease have similar signal intensity compared tosurrounding healthy tissue. In the case of magnetic resonance imaging,the imaging agent can also be referred to as a contrast agent.Lanthanide elements are known to be useful as contrast agents. Thelanthanide chemical elements comprises the fifteen metallic chemicalelements with atomic numbers 57 through 71, and include lanthanum,cerium, praseodymium, neodymium, promethium, samarium, europium,gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium,and lutetium. Preferred lanthanides include europium, gadolinium, andterbium. In order to more readily handle these rare earth metals, thelanthanides are preferably chelated. In some embodiments, the lanthanideselected for use as an imaging group is gadolinium, or more specificallygadolinium (Ill).

Methods of Imaging a Tissue Region Using a PSMA-Specific Imaging Agent

Another aspect of the invention provides a method for imaging prostatecancer in a tissue region of a subject that includes the steps of: (a)administering to the subject a detectably effective amount of aPSMA-specific imaging agent comprising a compound according to formulaI:

wherein S¹ is an organic spacer group having from 5 to 30 carbons, A isan amino acid forming a portion of a negatively charged peptideoligomer, n is from 3 to 6, S² is an organic spacer group having from 5to 15 carbons, and I is an imaging group, and pharmaceuticallyacceptable salts thereof; (b) allowing a sufficient amount of time forthe PSMA-specific imaging agent to enter the tissue region; and (c)performing imaging of the tissue region of the subject using an imagingdevice capable of detecting the imaging group. The imaging agent can beany of the imaging agents encompassed by formula I and/or describedherein. In some embodiments, the imaging device is a positron orsingle-photon emission tomography/computed tomography scanner, and theimaging group is a corresponding positron or single-photon emissiontomography imaging group. In other embodiments, the imaging device isnear-infrared imaging device, and the imaging group of the imaging agentis a near-infrared imaging group.

The present invention provides a method of generating an image of atissue region of a subject, by administering to the subject a detectablyeffective amount of a PSMA-specific imaging agent, and generating animage of the tissue region of the subject to which the imaging agent hasdistributed. In order to generate an image of the tissue region, it isnecessary for a detectably effective amount of imaging agent to reachthe tissue region of interest, but it is not necessary that the imagingagent be localized in this region alone. However, in some embodiments,the PSMA-specific imaging agents are targeted or administered locallysuch that they are present primarily in the tissue region of interest.Examples of images include two-dimensional cross-sectional views andthree dimensional images. In some embodiments, a computer is used toanalyze the data generated by the imaging agents in order to generate avisual image. The tissue region can be an organ of a subject such as theheart, lungs, or blood vessels. In other embodiments, the tissue regioncan be diseased tissue, or tissue that is suspected of being diseased,such as a tumor or atherosclerotic tissue. Examples of imaging methodsinclude optical imaging, computed tomography, positron emissiontomography, single photon emission computed tomography, and magneticresonance imaging.

Means of detecting labels in order to generate an image are well knownto those of skill in the art. Thus, for example, where the label is aradioactive label, means for detection include a scintillation counteror photographic film as in autoradiography. Where the label is afluorescent label, it may be detected by exciting the fluorochrome withthe appropriate wavelength of light and detecting the resultingfluorescence. The fluorescence may be detected visually, by means ofphotographic film, by the use of electronic detectors such as chargecoupled devices (CCDs) or photomultipliers and the like. Finally simplecolorimetric labels may be detected simply by observing the colorassociated with the label.

In some embodiments, the PSMA-specific imaging agent is detected usingoptical imaging. Optical imaging can be fast, safe, cost effective, andhighly sensitive. Scan times are on the order of seconds to minutes,there is no need for ionizing radiation, and the imaging systems can besimple to use. In addition, optical probes can be designed as dynamicmolecular imaging agents that may alter their reporting profiles in vivoto provide molecular and functional information in real time. In orderto achieve maximum penetration and sensitivity in vivo, the choice formost optical imaging in biological systems is within the red andnear-infrared (NIR) spectral region (600-900 nm), although otherwavelengths in the visible region can be used. In the NIR wavelengthrange, absorption by physiologically abundant absorbers such ashemoglobin or water, as well as tissue autofluorescence, is minimized.

Optical imaging includes all methods from direct visualization withoutuse of any device and use of devices such as various scopes, cathetersand optical imaging equipment, for example computer based hardware fortomographic presentations. The imaging agents are useful with opticalimaging modalities and measurement techniques including, but not limitedto: endoscopy; fluorescence endoscopy; luminescence imaging; timeresolved transmittance imaging; transmittance imaging; nonlinearmicroscopy; confocal imaging; acousto-optical imaging; photoacousticimaging; reflectance spectroscopy; spectroscopy; coherenceinterferometry; interferometry; optical coherence tomography; diffuseoptical tomography and fluorescence mediated molecular tomography(continuous wave, time domain frequency domain systems and earlyphoton), and measurement of light scattering, absorption, polarization,luminescence, fluorescence lifetime, quantum yield, and quenching.

In other embodiments, the PSMA-specific imaging agent can be detectedusing computed tomography. Computed tomography (CT) refers to adiagnostic imaging tool that computes multiple x-ray cross sections toproduce a cross-sectional view of the vascular system, organs, bones,and tissues. Positive emissions tomography and single photon emissioncomputed tomography refer to a diagnostic imaging tool in which thepatient receives a radioactive isotope by injection or ingestion whichthen computes multiple x-ray cross sections to produce a cross-sectionalview of the vascular system, organs, bones, and tissues to image theradioactive tracer. These radioactive isotopes are bound to compounds ordrugs that are injected into the body and enable study of the physiologyof normal and abnormal tissues.

Before or during these steps, an imaging device can be positioned aroundor in the vicinity of a subject (for example, an animal or a human) todetect signals emitted from the subject. The emitted signals can beprocessed to construct an image, for example, a tomographic image. Inaddition, the processed signals can be displayed as images either aloneor as fused (combined) images.

Another aspect of the invention provides a method of in vivo opticalimaging, wherein the illuminating and detecting steps are performedusing an endoscope, catheter, tomographic system, hand-held opticalimaging system, or an intraoperative microscope. In certain embodiments,the method is a method of in vivo imaging, wherein the presence,absence, or level of emitted signal is indicative of a disease state. Incertain embodiments, the method is a method of in vivo imaging, whereinthe method is used to detect and/or monitor a disease. In certainembodiments, the disease is cancer. Another aspect of the inventionprovides a method of in vivo imaging, wherein the signal emitted by theagent is used to construct an image. In other embodiments, the image isa tomographic image.

An imaging system useful in the practice of the invention typicallyincludes three basic components: (1) an appropriate source for inducingexcitation of the imaging agent, (2) a system for separating ordistinguishing emissions from the imaging agent, and (3) a detectionsystem. The detection system can be hand-held or incorporated into otheruseful imaging devices, such as intraoperative microscopes. Exemplarydetection systems include an endoscope, catheter, tomographic system,hand-held imaging system, or an intraoperative microscope.

A particularly useful emission gathering/image forming component is anendoscope. Endoscopic devices and techniques which have been used for invivo optical imaging of numerous tissues and organs. Other types ofemission gathering components are catheter-based devices, includingfiber optics devices. Such devices are particularly suitable forintravascular imaging.

Once the PSMA-specific imaging agent has been administered, a sufficientamount of time for the PSMA-specific imaging agent to enter the tissueregion. The time required for contrast agents to reach a tissue regionare known by those skilled in the art, and can be calculated based onavailable software, and vary depending on the injection site and theparticular tissue region.

The methods and compositions of the invention can be used to help aphysician or surgeon to identify and characterize areas of disease, suchas dysplasia and cancer, to distinguish diseased from normal tissues,such as detecting specific regions of prostate cancer within an organ orother tissues that are difficult to detect using ordinary imagingtechniques, and to further assess said tissues as candidates forparticular treatment regimens, or gauge the prognosis such as stagingthe cancer. The methods and compositions of the invention can also beused in the detection, characterization and/or determination of thelocalization of a disease, including early disease, the severity of adisease or a disease-associated condition, the staging of a disease,and/or monitoring a disease. The presence, absence, or level of anemitted signal can be indicative of a disease state.

The PSMA-specific imaging agents of the present invention can be used toimage a wide variety of different types of tissue regions. Examples ofdifferent types of tissue regions include portions of the cardiovascularsystem, such as the heart, blood vessels, carotid arteries, and theaorta; lung tissue, adipose tissue, brain tissue, hepatic tissue, renaltissue, and prostate tissue. All of these tissues can readily be imagedby injection of the PSMA-specific imaging agents. Note that whilePSMA-specific imaging agents are typically used to image a particulartissue region of interest, they can also be used to image an organ, or awhole body.

A prostate cancer tumor can be imaged using PSMA either at the prostate,or at other tissues subsequent to metastasis. Unlike many other cancers,prostate cancer is particularly difficult to detect using existingmolecular imaging tracers. There are several reasons for this, includingthe relatively slow growth and metabolic rate of prostate cancercompared to other malignancies as well as the small size of the organand proximity to the urinary bladder, into which mostradiopharmaceuticals are eventually excreted. Accordingly, in someembodiments, the tissue region is the prostate gland.

A tumor is an abnormal mass of tissue as a result of abnormal growth ordivision of cells caused by cancer. Tumors can occur in a variety ofdifferent types of tissue such as the breast, lung, brain, liver kidney,colon, and prostate, can be malignant or benign, and generally vary insize from about 1 cm to about 5 cm.

The imaging methods of the invention are suitable for imaging anyphysiological process or disease in which PSMA is involved. Typically,imaging methods are suitable for identification of areas of tissues ortargets which express high concentrations of PSMA. Typical applicationsinclude imaging malignant tumors or cancers that express PSMA, prostatecancer (including metastasized prostate cancer), and angiogenesis.Essentially all solid tumors express PSMA in the neovasculture.Therefore, methods of the present invention can be used to image nearlyall solid tumors including lung, renal cell, glioblastoma, pancreas,bladder, sarcoma, melanoma, breast, colon, germ cell, pheochromocytoma,esophageal and stomach. Also, certain benign lesions and tissuesincluding endometrium, schwannoma and Barrett's esophagus can be imagedaccording to the present invention. PSMA is frequently expressed inendothelial cells of capillary vessels in peritumoral and endotumoralareas of various malignancies such that compounds of the invention andmethods of imaging using same are suitable for imaging suchmalignancies.

Cancer Treatment Using PSMA-Specific Imaging Agents

PSMA-specific imaging agents can be used in a variety of differentmanners to carry out or assist in cancer treatment, and in particular inprostate cancer treatment. In one aspect, the PSMA-specific imagingagents are used to identify the location and/or severity of the cancer,after which the cancer is treated using a suitable method such assurgery or chemotherapy. In another aspect, the PSMA-specific imagingagents are modified by replacing the imaging group with a toxin group sothat the PSMA-specific imaging agents become PSMA-specific anticanceragents.

“Cancer” or “malignancy” are used as synonymous terms and refer to anyof a number of diseases that are characterized by uncontrolled, abnormalproliferation of cells, the ability of affected cells to spread locallyor through the bloodstream and lymphatic system to other parts of thebody (i.e., metastasize) as well as any of a number of characteristicstructural and/or molecular features. A “cancer cell” refers to a cellundergoing early, intermediate or advanced stages of multi-stepneoplastic progression. The features of early, intermediate and advancedstages of neoplastic progression have been described using microscopy.Cancer cells at each of the three stages of neoplastic progressiongenerally have abnormal karyotypes, including translocations, inversion,deletions, isochromosomes, monosomies, and extra chromosomes. Cancercells include “hyperplastic cells,” that is, cells in the early stagesof malignant progression, “dysplastic cells,” that is, cells in theintermediate stages of neoplastic progression, and “neoplastic cells,”that is, cells in the advanced stages of neoplastic progression.Examples of cancers are sarcoma, breast, lung, brain, bone, liver,kidney, colon, and prostate cancer.

In some embodiments, the method further includes the step of ablatingthe cancer. Ablating the cancer can be accomplished using a methodselected from the group consisting of cryoablation, thermal ablation,radiotherapy, chemotherapy, radiofrequency ablation, electroporation,alcohol ablation, high intensity focused ultrasound, photodynamictherapy, administration of monoclonal antibodies, and administration ofimmunotoxins.

In some embodiments, the step ablating the cancer includes administeringa therapeutically effective amount of an anticancer agent to thesubject. Examples of anticancer agents include angiogenesis inhibitorssuch as angiostatin K1-3, DL-α-difluoromethyl-ornithine, endostatin,fumagillin, genistein, minocycline, staurosporine, and (±)-thalidomide;DNA intercalating or cross-linking agents such as bleomycin,carboplatin, carmustine, chlorambucil, cyclophosphamide, cisplatin,melphalan, mitoxantrone, and oxaliplatin; DNA synthesis inhibitors suchas methotrexate, 3-Amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin,cytosine β-D-arabinofuranoside, 5-Fluoro-5′-deoxyuridine,5-Fluorouracil, gaciclovir, hydroxyurea, and mitomycin C; DNA-RNAtranscription regulators such as actinomycin D, daunorubicin,doxorubicin, homoharringtonine, and idarubicin; enzyme inhibitors suchas S(+)-camptothecin, curcumin, (−)-deguelin, 5,6-dichlorobenz-imidazole1-β-D-ribofuranoside, etoposine, formestane, fostriecin, hispidin,cyclocreatine, mevinolin, trichostatin A, tyrophostin AG 34, andtyrophostin AG 879, Gene Regulating agents such as5-aza-2′-deoxycitidine, 5-azacytidine, cholecalciferol,4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, alltrans-retinal, all trans retinoic acid, 9-cis-retinoic acid, retinol,tamoxifen, and troglitazone; Microtubule Inhibitors such as colchicine,dolostatin 15, nocodazole, paclitaxel, podophyllotoxin, rhizoxin,vinblastine, vincristine, vindesine, and vinorelbine; and various otherantitumor agents such as 17-(allylamino)-17-demethoxygeldanamycin,4-Amino-1,8-naphthalimide, apigenin, brefeldin A, cimetidine,dichloromethylene-diphosphonic acid, leuprolide,luteinizing-hormone-releasing hormone, pifithrin-, rapamycin,thapsigargin, and bikunin.

Another method of ablating cancer such as prostate cancer that has beendetected using a PSMA-specific imaging agent is to conducting surgery toremove the cancer tissue (e.g., prostate cancer tissue) from thesubject. The main type of surgery for prostate cancer is known as aradical prostatectomy. Radical prostatectomy involves removing theentire prostate gland plus some of the tissue around it, including theseminal vesicles. Examples of types of radical prostatectomy includeradical retropubic prostatectomy, radical perineal prostatectomy, andlaparoscopic radical prostatectomy.

In some embodiments, the surgery used to remove the cancer is roboticsurgery that is guided by use of the PSMA-specific imaging agent. Forexample, the robotic surgery can be near-infrared fluorescence-guidedrobotic surgery. A preferred type of robotic surgery is robotic-assistedlaparoscopic radical prostatectomy (RALRP). A robotic surgery system forperforming robotic surgery with a surgery robot using guiding images ofa part to be operated on, the robotic surgery system comprising: anendoscope apparatus for capturing medical images of a predeterminedorgan in a body to be examined; a non-endoscopic apparatus including atleast one of an ultrasound apparatus, a computed tomography (CT)apparatus, a magnetic resonance imaging (MRI) apparatus, and a positronemission tomography (PET) apparatus for capturing medical images of thepredetermined organ; a medical image processing apparatus for acquiringthe medical images captured using the plurality of multi-modal medicalimage capturing apparatuses, extracting surface information of thepredetermined organ, which is included in each of the medical images,from each of the medical images, mapping each of the medical imagesusing the extracted surface information, and generating a synthesisimage in which the medical images have been registered, based on themapping result; a display apparatus for displaying the generatedsynthesis image; and the surgery robot for performing a robotic surgery.See for example US Patent Publications 2013/0035583 and 2013/0211420,the disclosures of which are incorporated herein by reference.

In some embodiments, PSMA-specific anticancer agents in which theimaging group has been replaced with a toxin are used to treat prostatecancer. In other embodiments, the PSMA-specific anticancer agents areused to treat metastatic cancer which has spread to one or more sitesbeyond the initial point where cancer has occurred. As noted herein,because PSMA occurs in a variety of different types of cancer, thePSMA-specific anticancer agents can be used to treat cancer other thanprostate cancer.

Accordingly, in one aspect, the invention provide methods of treatingtumors by administering to a subject a therapeutically effective amountof a PSMA-specific anticancer agent comprising a therapeuticallyeffective toxin such as a radioisotope. In certain embodiments, thetumor cells may express PSMA, such as prostate tumor cells ormetastasized prostate tumor cells. In other embodiments, a tumor may betreated by targeting adjacent or nearby cells which express PSMA. Forexample, vascular cells undergoing angiogenesis associated with a tumormay be targeted. Essentially all solid tumors express PSMA in theneovasculture. Therefore, methods of the present invention can be usedto treat nearly all solid tumors including lung, renal cell,glioblastoma, pancreas, bladder, sarcoma, melanoma, breast, colon, germcell, pheochromocytoma, esophageal and stomach. Also, certain benignlesions and tissues including endometrium, schwannoma and Barrett'sesophagus can be treated according to the present invention. Examples oftherapeutically effective radioisotopes include ¹³¹I and ²¹¹At.

Administration and Formulation of PSMA-Specific Imaging and AnticancerAgents

In some embodiments, the PSMA-specific imaging agent is administered ina pharmaceutically acceptable carrier, e.g., an aqueous carrier. Avariety of carriers can be used, e.g., buffered saline and the like.These solutions are sterile and generally free of undesirable matter.The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, for example, sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate and the like. The concentration ofactive agent in these formulations can vary widely, and will be selectedprimarily based on fluid volumes, viscosities, body weight and the likein accordance with the particular mode of administration and imagingmodality selected.

Administration of the PSMA-specific imaging agent for in vivo imaging ofa tissue, an organ or a full body can include a) providing apharmaceutical formulation comprising the imaging agent of the inventionand a pharmaceutically acceptable excipient, wherein the imaging agentis formed according to any of the above described embodiments, andwherein the formulation is suitable for administration as aPSMA-specific imaging agent and the imaging agent is present in adetectably effective amount; b) providing an imaging device (i.e., anoptical imaging device); c) administering the pharmaceutical formulationin an amount sufficient to generate the tissue or body image; and d)imaging the distribution of the pharmaceutical formulation of step a)with the imaging device, thereby imaging the tissue, organ or body.

The pharmaceutical formulations of the invention can be administered ina variety of unit dosage forms, depending upon the particular tissue orcancer to be imaged, the general medical condition of each patient, themethod of administration, and the like. Details on dosages are welldescribed on the scientific and patent literature. The exact amount andconcentration of PSMA-specific imaging agent or pharmaceutical of theinvention and the amount of formulation in a given dose, or the“detectably effective dose” can be routinely determined by, e.g. theclinician. The “dosing regimen” will depend upon a variety of factors,e.g. whether the tissue region or tumor to be imaged is disseminated orlocal, the general state of the patient's health, age and the like.Using guidelines describing alternative dosing regimens, e.g. from theuse of other imaging agents, the skilled artisan can determine byroutine trials optimal effective concentrations of pharmaceuticalcompositions of the invention.

The pharmaceutical compositions of the invention can be delivered by anymeans known in the art systematically (e.g. intravenously), regionallyor locally (e.g. intra- or peritumoral or intracystic injection, e.g. toimage bladder cancer) by e.g. intraarterial, intratumoral, intravenous(iv), parenteral, intrapneural cavity, topical, oral or localadministration, as sub-cutaneous intra-zacheral (e.g. by aerosol) ortransmucosal (e.g. voccal, bladder, vaginal, uterine, rectal, nasal,mucosal), intra-tumoral (e.g. transdermal application or localinjection). For example, intra-arterial injections can be used to have a“regional effect”, e.g. to focus on a specific organ (e.g. brain, liver,spleen, lungs).

Preparation of the Compounds

PSMA-specific imaging agents may be synthesized by synthetic routes thatinclude processes similar to those well known in the chemical arts,particularly in light of the description contained herein. The startingmaterials are generally available from commercial sources such asAldrich Chemicals (Milwaukee, Wis., USA) or are readily prepared usingmethods well known to those skilled in the art (e.g., prepared bymethods generally described in Louis F. Fieser and Mary Fieser, Reagentsfor Organic Synthesis, v. 1-19, Wiley, New York, (1967-1999 ed.); AlanR. Katritsky, Otto Meth-Cohn, Charles W. Rees, Comprehensive OrganicFunctional Group Transformations, v 1-6, Pergamon Press, Oxford,England, (1995); Barry M. Trost and Ian Fleming, Comprehensive OrganicSynthesis, v. 1-8, Pergamon Press, Oxford, England, (1991); orBeilsteins Handbuch der organischen Chemie, 4, Aufl. Ed.Springer-Verlag, Berlin, Germany, including supplements (also availablevia the Beilstein online database)).

Those skilled in the art will appreciate that synthetic routes otherthan those described in the examples herein may be used to synthesizethe compounds of the invention. Although specific starting materials andreagents are depicted in the reaction schemes and discussed below, otherstarting materials and reagents can be easily substituted to provide avariety of derivatives and/or reaction conditions. In addition, many ofthe compounds prepared by the methods described below can be furthermodified in light of this disclosure using conventional methods wellknown to those skilled in the art.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1 Improving the Biodistribution of PSMA-TargetingTracers with a Highly Negatively Charged Linker

Prostate specific membrane antigen (PSMA) is overexpressed in prostatecancer and in tumor vasculature. Small molecule based inhibitors of PSMAhave promised to provide sensitive detection of primary and metastaticprostate tumors. Although significant progress has been made, many ofthe radiolabeled imaging agents exhibit non-specific background binding.Prevailing tracer designs focus on high affinity urea-based inhibitorswith strategically placed hydrophobic patches that interact favorablywith the substrate tunnel of PSMA. The inventors hypothesized that anovel PSMA inhibitor design incorporating highly negatively chargedlinkers may minimize non-specific binding and decrease overallbackground.

Materials and Methods:

(S)-2-(3-((S)-5-amino-1-carboxypentyl)ureido)pentanedioic acid(Cys-C(O)-Glu) was custom-made by Bachem Bioscience Inc.H-Glu(OtBu)-OtBu is from Bachem (King of Prussia, Pa.).N—[N—[(S)-1,3-dicarboxypropyl]carbamoyl]-S-[³H]-methyl-L-cysteine wascustom made by GE Healthcare Life Sciences. Succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) was purchased fromThermo Fisher Scientific, Rockford, Ill. Rink Amid MBHA resin, andFmoc-(D)Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OHwere purchased from Peptides International Inc, Louisville Ky. TheFmoc-Glu-OtBu was from Novabiochem, Merck KGaA, Darmstadt, Germany. Allthe other chemicals were purchased from Sigma-Aldrich Inc., St. Louis,Mo. High resolution matrix-assisted laser desorption/ionization mass(MALDI-MS) spectra were obtained from an Applied Biosystems 4800MALDI/TOF Analyzer in positive ion mode.

High Performance Liquid Chromatography (HPLC) was performed on aShimadzu HPLC system equipped with a SPD-20V prominence UV/visibledetector and monitored at a wavelength of 260 nm. Preparative HPLC wasachieved using a SymmetryPrep™ C18 column (100 mm×19 mm×5 m, WatersCorporation, Milford, Mass., USA) at a flow rate of 3.0 ml/min.Analytical HPLC was performed using an analytical Symmetry C18 column(150 mm×4.6 mm×5 m, Waters Corporation, Milford, Mass., USA). Flow rateis 1.0 mL/min unless otherwise specified.

Synthesis of Compound 1 (ZJ-MCC-Ahx-dEdEdEG): The peptideFmoc-Ahx-dGlu-dGlu-dGlu-G was assembled on a Wang resin. The threeglutamates (dGlu) are of D-isoform. Peptide synthesis was carried outmanually by Fmoc chemistry with HCTU(2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate) activation. Generally, peptides were synthesized ata 0.01 mmol scale starting from the C-terminal amino acid on solidsupport. Fmoc-deprotection at each cycle was carried out using 20%piperidine in DMF. Coupling reactions were carried out using 3.3 eq. ofFmoc-amino acids in DMF activated with 3.3 eq. of HCTU and 5 equivalentsof diisopropylethylamine (DIPEA) in DMF. These steps were repeated eachtime with an amino acid added. After the peptide sequence was built onthe resin, the Fmoc group of the N-terminal amino acid was deprotected.Coupling of 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) to theN-terminal amine group was achieved with 3.3 equivalents of SMCC in DMF.Coupling of Cys-C(O)-Glu was performed using 3.3 equivalents ofCys-C(O)-Glu in DMF after coupling SMCC to the peptide. The finalpeptide resin was washed with DMF and then dichloromethane and dried.Cleavage and deprotection were carried out usingTFA/water/triisopropylsilane (950:25:25) for 1 h, the resin was removedby filtration and washed with TFA. The combined filtrate was dried undernitrogen. The synthesized peptide was precipitated by the addition ofdiethyl ether and collected by centrifugation. The cleaved peptide waspurified by preparative HPLC. The products were ascertained by highresolution matrix-assisted laser desorption/ionization mass (MALDI-MS)spectra. Then Fmoc was deprotected followed by coupling of SMCC andCys-C(O)-Glu. The product has retention time of 11.9 minutes onanalytical HPLC with 0-55% gradient over 45 minutes (flow rate 1 ml/min;A: 10 mM triethylammonium acetate TEAA, pH 7.0; B was acetonitrile.) Themass was verified by MALDI/TOF mass spectrometry—Calculated: 1088.4(C44H64N8O22S). Found m/z: 1089.4 (M+1).

Compound 2—(ZJ-MCC-Ahx-YYYG): The peptide Fmoc-Ahx-Tyr-Tyr-Tyr-Gly wasassembled on Wang resin. Then Fmoc was deprotected followed by couplingof SMCC and Cys-C(O)-Glu as described for compound 1. The product hasretention time of 14.9 minutes on analytical HPLC with 0-55% gradientover 45 minutes (flow rate 1 ml/min; A: 10 mM triethylammonium acetateTEAA, pH 7.0; B was acetonitrile.) The mass was verified by MALDI/TOFmass spectrometry—Calculated: 1190.5 (C56H70N8O19S). Found m/z: 1191.4(M+1).

Synthesis of Compound 3: E′E-Amc-Ahx-dEdEdEGYGGGC-NH₂

Fmoc-′E-Amc-Ahx-dGlu-dGlu-dGlu-Gly-Tyr-Gly-Gly-Gly-Cys-NH₂ was assembledon the resin using standard Fmoc peptide synthesis. Fmoc-′E stands forFmoc(Glu)-OtBu where the gamma-carboxyl group is unprotected. Afterremoval of the last Fmoc on the assembled peptide, the resin is washedwith DMF, methanol and chloroform. Then, a chloroform solutioncontaining a 5-fold excess of H-Glu(OtBu)-OtBu mixed with 2.5 eq (withrespect to H-Glu(OtBu)-OtBu) of diisopropylethylamine was prepared. Thesolution was then added slowly to 0.25 eq (with respect toH-Glu(OtBu)-OtBu)triphosgene over 10 minutes at room temperature. Thepresumed product of this reaction is an isocyanate derivative ofH-Glu(OtBu)-OtBu. After a 15 minute incubation to allow for isocyanateformation, the reaction is mixed with the′E-Amc-Ahx-Glu-Glu-Glu-Gly-Tyr-Gly-Gly-Gly-Cys-NH₂ on a rink amide resinpre-swollen in chloroform with 2.5 eq of diisopropylethylamine. After 30minutes of mixing, a Ninhydrin test was administered to test forresidual free-amine on the resin. Once the reaction is complete, theresin is washed and the complete peptide product is cleaved. The productelutes at 12.4 minutes on analytical HPLC column with a 10%-95% gradientin 40 minutes (flow rate 0.8 ml/min; A: water with 0.1% TFA; B:acetonitrile). The mass was verified by MALDI/TOF massspectrometry—Calculated: 1452.6 (C60H88N14O26S). found m/z: 1453.4(M+1).

[^(99m)Tc]3: Tc99m Labeling of Compound 3

The procedure was modified from that used by Tolmacheva and coworkersfor Affibody labeling. Ahlgren et al., Nuclear medicine and biology;37(5):539-546 (2010). An aqueous solution of peptide compound 6 (20 μL0.5 mM) was first mixed with 10 μL of EDTA (10 mg/mL), 10 μL of sodiumgluconate (375 mg/mL) and 10 μL stannous chloride (7.5 mg/mL in 10 mMHCl). Then 10-12 mCi of freshly eluted ^(99m)Tc pertechnetate was addedand the solution was heated to 95° C. for 1 hour. The labeling mixturewas loaded on a SepPak cartridge, and washed with 3 mL of saline twice.The radiolabeled [^(99m)Tc]3 is then eluted with 1 mL of 100% ethanol.The ethanol is evaporated and the remaining solid is dissolved in salineand analyzed by TLC. Radiochemical purity by TLC is typically 95%.

Synthesis of Compound 4: E′E-Amc-Ahx-dEdEdEYK(Bn-NOTA)-NH₂

Fmoc-′E-Amc-Ahx-dGlu-dGlu-dGlu-Tyr-Lys-NH₂ was assembled on the resinusing standard Fmoc peptide synthesis. The glutamates (dGlu) areD-isomers. Fmoc-′E stands for Fmoc(Glu)-OtBu where the gamma-carboxylgroup is unprotected. The last Fmoc on the assembled peptide is thenremoved by 20% piperidine. Then a chloroform solution containing 5 eq.of H-Glu(OtBu)-OtBu mixed with 2.5 eq (with respect to H-Glu(OtBu)-OtBu)of diisopropylethylamine was prepared. The solution was then addedslowly to 0.25 eq (with respect to H-Glu(OtBu)-OtBu)triphosgene inchloroform over 10 minutes at room temperature. After a 15 minuteincubation to allow for isocyanate formation, the reaction is mixed withthe ′E-Amc-Ahx-Glu-Glu-Glu-Gly-Tyr-Gly-Gly-Gly-Cys-NH₂ on a rink amideresin pre-swollen in chloroform with 2.5 eq of diisopropylethylamine.After 30 minutes of mixing, a Ninhydrin test was administered to testfor residual free-amine on the resin. The reaction was repeated ifneeded. Once the reaction is complete, the resin is washed and thecomplete peptide product is cleaved. To couple the purified peptideE′EAmc-Ahx-EEEYK(Bn-NOTA)-NH₂ with SCN-Bn-NOTA (Macrocyclics),E′EAmc-Ahx-dEdEdEYK was dissolved in DMF at a concentration of 25 mg/mLand an equimolar amount of SCN-Bn-NOTA was dissolved in DMSO at aconcentration of 200 mg/mL. After mixing the above DMF and DMSOsolutions of the reactants, DIPEA was added to concentration of 2% v/v.The reaction was monitored by HPLC and allowed to proceed up to 2 hours.Then, glacial acetic acid equivolume to DIPEA is added to stop thereaction. The final product wasE′EAmc-Ahx-dGlu-dGlu-dGlu-Tyr-Lys(Bn-NOTA)-NH₂ (compound 4) The productelutes at 14.8 min on an analytical column with a 10%-90% gradient in 45minutes with a flow rate of 0.8 ml/min (A: water with 0.1% TFA; B:acetonitrile). The mass was verified by MALDI/TOF massspectrometry—Calculated: 1699.7. found m/z: 1700.7 (M+1).

Synthesis of Compound 5: E′E-Ahx-EEEYK(Bn-NOTA)-NH₂

Fmoc-′E-Ahx-Glu-Glu-Glu-Tyr-Lys-NH₂ was assembled on the resin usingstandard Fmoc peptide synthesis. The Glutamates are L-isomers. Fmoc-′Estands for Fmoc(Glu)-OtBu where the gamma-carboxyl group is unprotected.The last Fmoc on the assembled peptide is then removed by 20%piperidine. Then, a chloroform solution containing 5 eq ofH-Glu(OtBu)-OtBu mixed with 2.5 eq (with respect to H-Glu(OtBu)-OtBu) ofdiisopropylethylamine was prepared. The solution is then added slowly to0.25 eq. (with respect to H-Glu(OtBu)-OtBu)triphosgene in chloroformover 10 minutes at room temperature. After a 15 minute incubation toallow for isocyanate formation, the reaction is mixed with the′E-Amc-Ahx-Glu-Glu-Glu-Gly-Tyr-Gly-Gly-Gly-Cys-NH₂ on a rink amide resinpre-swollen in chloroform with 2.5 eq. of diisopropylethylamine. After30 minutes of mixing, a Ninhydrin test was administered to test forresidual free-amine on the resin. Once the reaction was complete, theresin was washed and the complete peptide product was cleaved. Theproduct was then purified by HPLC with acetonitrile/water with 0.1% TFA.Coupling of the purified peptide E′E-Ahx-EEEYK(Bn-NOTA)-NH₂ withSCN-Bn-NOTA (Macrocyclics) was performed with a method derived from Langet. al. Lang et al., Bioconjug Chem; 22(12): 2415-2422 (2011). Briefly,E′E-Ahx-EEEYK was dissolved in DMF at concentration of 25 mg/mL and anequimolar amount of SCN-Bn-NOTA was dissolved in DMSO at a concentrationof 200 mg/mL. After mixing the above DMF and DMSO solutions of thereactants, DIPEA was added to concentration of 2% v/v. The reaction wasmonitored by HPLC and allowed to proceed up to 2 hours. Then, glacialacetic acid equivolume to DIPEA was added to stop the reaction. Thefinal product E′E-Ahx-Glu-Glu-Glu-Tyr-Lys(Bn-NOTA)-NH₂ (compound 5)elutes at 13.4 minutes on a (10% B-95% B HPLC gradient in 40 minuteswith a flow rate of 0.8 ml/min on an analytical column. (A: water with0.1% TFA, B: acetonitrile) The mass was verified by MALDI/TOF massspectrometry—Calculated: 1560.6. found m/z: 1561.7 (M+1).

¹⁸F Labeling of Compound 5

1 Ci of ¹⁸F sodium fluoride in water was purchased from Siemens PETNETSolutions in Cleveland, Ohio. The activity was loaded onto a QMA columnand eluted with a 200 μL fraction of saline. 15 μL of the fractioncontaining the highest radioactivity was added to a solution containing6 μL of 2 mM AlCl₃ in 100 mM NaOAc, 2 μL of ascorbic acid (50 mg/mL inwater), 20 nmol of E′E-Ahx-EEEYK(Bn-NOTA)-NH₂ (compound 5) dissolved in20 μL of water and 4 μL of 1 M sodium acetate pH 4.1. The total volumeof the solution was 40 μL. The solution was heated on a 105° C. heatingblock for 15 minutes. The reaction mixture was purified with analyticalHPLC using 15% B for 5 minutes followed by 20% B (flow rate 1 min/min,A: water 0.1% TFA; B: acetonitrile). Un-reacted compound 8 elutes at13.6 minutes. Radiolabeled [Al¹⁸F]5 elutes at 10.7 minutes. The purifiedfraction is diluted ⅓ with mobile phase (0.1% TFA water) and loaded ontoSepPak. The SepPak column was washed with 3 mL water and eluted with 1mL of 90% ethanol and 10% mobile phase. The typical decay-correctedradiochemical yield is 10%. The ethanol and TFA in the collectedfraction were evaporated and normal saline was added to titrate thedose.

Cell Culture

The prostate cancer cell line LNCaP (PSMA-positive) was obtained fromAmerican Type Culture Collection (Manassas, Va.). Retrovirallytransformed PSMA positive PC3-PIP cells and transfection controlledPC3-flu cells were obtained from Dr. Michel Sadelain (Laboratory of GeneTransfer and Gene Expression, Gene Transfer and Somatic Cell EngineeringFacility, Memorial-Sloan Kettering Cancer Center, New York, N.Y.). Cellswere grown at 37° C. and 5% CO₂ under a humidified atmosphere. Cellswere maintained in RPMI1640 medium supplemented with 2 mM L-glutamineand 10% Fetal Bovine Serum.

Competitive Binding Assay

Briefly, LNCaP or PC3-PIP cells (5×10⁵) were incubated with differentconcentrations of ligands in the presence of 12 nMN—[N—[(S)-1,3-dicarboxypropyl]carbamoyl]-S-[³H]-methyl-L-cysteine in atotal volume of 300 μL for 1 hour at 37° C. The mixture was centrifugedat 3,000 g for 5 min at 4° C., then washed three times with 500 μL ofcold PBS. Finally, 4 mL of EcoLume™ cocktail (MP Biomedicals) was added,and radioactivity was counted by scintillation counter. Theconcentration required to inhibit 50% of binding was determined (IC₅₀)by GraphPad Prism 3.0.

SPECT/CT and Biodistribution Study of [^(99m)Tc]3

Male NOD scid gamma (NSG) mice bearing PC3-PIP (PSMA+) and PC3-flu(PSMA−) tumor xenographs were used for imaging. The mice were injectedwith 3 mCi of [^(99m)Tc]3. Four hours after tracer injection, SPECT/CTimages were acquired using a high-resolution multi-pinhole SPECT withcollimation insert with a clinical SPECT/CT scanner (Symbia T6, SiemensMolecular Imaging). DiFilippo, F. P. Phys Med Biol 53(15): 4185-4201(2008). For the biodistribution study without imaging, male NSG micebearing two PC3-PIP tumors were injected with 50-100 Ci of [^(99m)Tc]3.Mice were sacrificed at 2 hours, 4 hours and 6 hours after injection.After thoracotomy, blood was first withdrawn from the heart, then theorgans were resected en-block. The activities were counted with a PerkinElmer 2480 Automatic Gamma Counter. The decay corrected percent injecteddose per gram of organ weight (wet) (% ID/g) was calculated.

Positron emission tomography and biodistribution study of [Al¹⁸F]5: MaleNSG mice at least 6 weeks old were subcutaneously implanted with twohuman PC3-PIP (PSMA+) tumor xenographs. When one of the tumors reached0.5 mL in volume, imaging and biodistribution studies were conducted.For imaging, 0.3-0.4 mCi of [Al¹⁸F]5 was injected via the tail vein. Themouse was anesthetized with isoflurane and imaged at 1 hour postinjection on a clinical PET/CT scanner (Biograph mCT, Siemens MolecularImaging, Hoffman Estates, Ill.) using super-resolution sampling anditerative deconvolution processing. For the biodistribution studywithout imaging, 50-150 μCi of [Al¹⁸F]5 was injected in PC3-PIP tumorbearing mice which were sacrificed at one and two hours post injectionfor biodistribution measurements.

Results:

Binding affinities of new compounds to PSMA. The equilibrium competitivebinding studies of all the compounds synthesized are summarized inTable 1. All of the tested compounds exhibited similar affinity as shownin Table 1.

TABLE 1 IC₅₀ of compounds. 95% confidence intervals are reported inparentheses. IC₅₀ [nM] ZJ-MCC-Ahx-dEdEdEG (Compound 1)  2.2 (0.83-5.5)ZJ-MCC-Ahx-YYYG (Compound 2) 2.5 (1.1-5.6) E′EAmc-Ahx-dEdEdEGYGGGC-NH₂(Compound 3)  7.0 (0.72-14) E′EAmc-Ahx-dEdEdEYK(Bn-NOTA)-NH₂ (Compound4.6 (1.6-6.1) 4) E′E-Ahx-EEEYK(Bn-NOTA)-NH₂ (Compound 5)  4.3 (0.78-14)ZJ24 (the “parent” di-amino acid urea ligand (S)-2-(3- 4.3 (2.1-9.1)((S)-5-amino-1-carboxypentyl)ureido)pentanedioic acid)

Synthesis of Tc99m Tracer

Using RBI-1033 as a template, the inventors started their iterativere-design by considering peptide mimics of the 2-5 Å moiety (FIG. 1).One possible type of interaction between the 2-5 Å moiety and the tunnelmay be a charge-charge interaction through the negatively chargedphosphate backbone and the interaction of aromatic amino acid residuesof PSMA with the adenine moiety. The inventors hypothesized thatZJ-MCC-Ahx-dEdEdEG (compound 1), which has three glutamic acids, canmimic the negative charge of the phosphate backbone of 2-5 Å.ZJ-MCC-Ahx-YYYG (compound 2) was also synthesized, which has tyrosineresidues, to mimic the potential hydrogen bonding and pi-pi interactionof the nucleoside component of the 2-5 Å moiety of RBI-1033. Bothdemonstrated affinity comparable to the parent urea ligandMeS-Cys-C(O)-Glu (ZJ24) (Table 1).

The inventors decided to continue the iterative design process usingZJ-MCC-Ahx-dEdEdEG (compound 1) as a template. To streamline thesynthesis and to decrease cost, the SMCC based linker was modifiedbetween the tri-glutamate and the urea moiety. The thioether bond wasreplaced and the succinimide with a glutamyl side chain that iscovalently linked to N-aminomethyl cyclohexanoic acid (Amc) via apeptide bond (FIG. 1). Compared to SMCC, this arrangement reduces sterichindrance by eliminating one side of the succinimide and shortens thelinker by one carbon-carbon bond. The changes also allow the entirecompound to be synthesized on the solid phase. To make a ^(99m)Tcbinding tracer from the peptide framework, a Gly-Tyr spacer was addedbetween the triglutamate moiety and an N3S1 technetium chelateconsisting of a Gly-Gly-Gly-Cys peptide (FIG. 1). The modificationsresulted in E′EAmc-Ahx-dEdEdEGYGGGC (compound 3). The inventors used the280 nm absorption of the tyrosine residue for concentration measurementsto standardize the preparation. The affinity of the unlabeled compoundis equivalent to compound 1 (Table 1).

SPECT/CT Imaging and Biodistribution Study of^(99m)Tc-E′EAmc-Ahx-dEdEdEGYGGGC ([^(99m)Tc]3)

Labeling with technetium typically yields 95% radiochemical purity afterSepPak purification. The imaging studies (FIG. 2) demonstrated specifictargeting of PSMA+PC3-PIP tumors with no significant binding to PC3-flutumors. The kidneys and bladder are the most prominent organs on theimages, because PSMA is expressed naturally in mouse kidneys and becauseit is a renally excreted compound. The amount of tracer in the measuredtissue and organs did not change significantly from 2 hours to 6 hours.Tumor uptake is 6.0±1.6% at 2 hours, 6.0±2.2% at 4 hours and 5.7%±2.5%at 6 hours (Table 2). There is a small amount of radioactivity in thethyroid (0.3% ID/g) and the stomach (0.2% ID/g) which is indicative ofthe presence of ^(99m)Tc-pertechnetate. This is likely related toresidual pertechnetate after cartridge purification and dissociation oftechnetium from the chelate. Attempts were made to use HPLC purificationwith C18 columns, however, the ^(99m)Tc-labeled complex is not stableunder solvent conditions for HPLC, which contains 0.1% trifluoroaceticacid.

TABLE 2 Biodistribution of [^(99m)Tc]3 (Unit: % ID/g: N = 6 for tumor, N= 3 for other organs) 2-hour 4-hour 6-hour Tumor 6.03% ± 1.63% 5.95% ±2.23% 5.71% ± 2.53% (PC3-PIP) Thyroid 1.47% ± 0.77% 0.28% ± 0.14% 0.33%± 0.30% Heart 0.21% ± 0.02% 0.04% ± 0.01% 0.03% ± 0.01% Liver 0.28% ±0.07% 0.09% ± 0.03% 0.08% ± 0.04% Bone(femur) 0.12% ± 0.04% 0.07% ±0.07% 0.06% ± 0.08% Stomach 1.00% ± 0.33% 0.19% ± 0.12% 0.20% ± 0.17%Blood 0.27% ± 0.08% 0.04% ± 0.00% 0.05% ± 0.03% Small Intestine 0.24% ±0.11% 0.06% ± 0.00% 0.06% ± 0.00% Large Intestine 0.60% ± 0.36% 0.82% ±0.23% 0.57% ± 0.34% Kidney 60.58% ± 18.10% 16.06% ± 20.05% 17.43% ±18.92% Spleen 1.14% ± 0.30% 0.18% ± 0.11% 0.20% ± 0.09% Pancreas 0.18% ±0.04% 0.04% ± 0.02% 0.03% ± 0.03% Lung 0.35% ± 0.06% 0.09% ± 0.05% 0.09%± 0.05% Muscle 0.11% ± 0.01% 0.04% ± 0.03% 0.03% ± 0.04% Tumor:bone 50:1 85:1  95:1 Tumor:blood 22:1 149:1 114:1 Tumor:muscle 55:1 149:1 190:1Tumor:liver 22:1  66:1  71:1

Synthesis of ¹⁸F Labeled PSMA Targeting Tracer

To accurately estimate the biodistribution of the highly negativelycharged tracer, high radiochemical purity was needed. Since preparationof [^(99m)Tc]3 without ^(99m)Tc-pertechnetate was not possible, theinventors searched for another radiolabeling strategy.Aluminum-fluoride-NOTA complex based ¹⁸F labeled peptides, firstdemonstrated by McBride et. al., are stable enough for purification withHPLC with 0.1% TFA in the solvent. McBride et al., J Nucl Med;50(6):991-998 (2009). The labeled complex also results in an easilyprepared ¹⁸F labeled tracer that can be used for positron emissiontomography. To incorporate a NOTA chelate, the sequenceGly-Tyr-Gly-Gly-Gly-Cys associated with the N3S1 chelate for ^(99m)Tcwas replaced with a simple Tyr-Lys. The lysine is used for coupling witha commercially available activated NOTA chelate. The resulting Compound4: E′EAmc-Ahx-dEdEdEYK(Bn-NOTA) can be labeled after 15 minutes ofheating with 18F fluoride and aluminum chloride in acetate buffer. Theresulting radioactive complex ¹⁸Faluminum-fluoride-E′EAmc-Ahx-dEdEdEYK(Bn-NOTA) can be easily purifiedwith HPLC. To further reduce hydrophobicity, the Amc moiety waseliminated from the linker of compound 4. The purpose of the Amc was tomimic the cyclohexane portion of the linker in RBI-1033. The inventorsalso changed the glutamate from D-glutamate to L-glutamate to reduce thecost of synthesis. They previously demonstrated that one can switch theconformation of the three glutamates from the D-isomer to the L-isomerwithout change in affinity and imaging characteristics of NIR PSMAtargeting tracers. The resulting compound 5 (E′E-Ahx-EEEYK(Bn-NOTA)),which is cheaper and more convenient to produce, has the same affinityfor PSMA as compound 4 (E′E-Amc-AhxdEdEdEYK(Bn-NOTA)). The inventorsproceeded to perform PET imaging and a biodistribution study with the¹⁸F labeled aluminum-fluoride-compound 5 ([Al¹⁸F]5).

The one-hour post injection images (FIG. 3) demonstrate uptake in thePC3-PIP tumor and renal parenchyma and accumulation in the renalcollecting systems and bladder. No significant tracer activity is seenin other tissue. The biodistribution data for [Al¹⁸F]5 indicated veryfast renal excretion with the amount of radioactivity in blood, bone andmuscle dropping to 0.04% ID/g, 0.1% ID/g and 0.01% ID/g respectively at2 hours post injection (Table 3). The tumor uptake was 4.6% ID/g at 2hours. The thyroid and stomach uptake that was seen with [^(99m)Tc]3were reduced to background as expected. The minimal bone uptakeindicated no significant dissociation of ¹⁸F-fluoride from the complex.

TABLE 3 Preliminary 1-hour and 2-hour biodistribution of F-18 labeled[Al¹⁸F]5 (Unit: % ID/g) 1 hour (n = 4 for 2 hour (n = 6 for tumor, n = 2for organ) tumor; n = 3 for organ) Tumor 6.91% ± 1.44% 4.58% ± 0.55%(PC3PIP) Thyroid 0.11% ± 0.02% 0.05% ± 0.00% Heart 0.14% ± 0.11% 0.04% ±0.01% Liver 0.41% ± 0.10% 0.17% ± 0.03% Bone 0.39% ± 0.28% 0.10% ± 0.02%Stomach 0.10% ± 0.03% 0.03% ± 0.02% Blood 0.18% ± 0.09% 0.04% ± 0.01%Small Intestine 0.43% ± 0.51% 0.18% ± 0.02% Kidney 16.82% ± 5.60%  5.96%± 2.17% Spleen 0.29% ± 0.14% 0.10% ± 0.04% Lung 0.21% ± 0.05% 0.07% ±0.04% Muscle 0.19% ± 0.12% 0.01% ± 0.00% Tumor:bone 18:1 46:1Tumor:blood 38:1 115:1  Tumor:muscle 36:1 458:1  Tumor:liver 17:1 27:1Tumor:spleen 24:1 46:1

Discussion

Affinity vs. Background

There is growing recognition of the problem of nonspecific backgroundbinding in the initial human trials of PET and SPECT tracers targetingPSMA. Reske et al., Eur J Nucl Med Mol Imaging; 40(6):969-970 (2013).Afshar-Oromieh et al., Eur J Nucl Med Mol Imaging; 40(6):971-972 (2013).

Among the published images, there appear to be two general patterns ofnonspecific uptake. One is in the salivary glands, lacrimal glands andliver such as those demonstrated by ¹²³I-MIP-1072, ¹²³I-MIP-1095,Glu-NH—CO—NH-Lys(Ahx)-[⁶⁸Ga(HBED-CC)]. Barrett et al., J Nucl Med;54(3):380-387 (2013). The other is elevated blood pool backgroundactivity, such that exhibited by ¹⁸F-DCFBC and ⁶⁸Ga-DOTA-DUPA-Pep. Choet al., J Nucl Med; 53(12):1883-1891 (2013). There is no clearcorrelation between molecular structure and patterns of nonspecificactivity. However, hydrophobic patches within the molecules could be thereason for nonspecific binding. The existing literature emphasizes theconstruction of high affinity PSMA ligands by judicious placement ofhydrophobic moieties. Barinka et al. uncovered an accessory hydrophobicpocket near the S1 site of the PSMA enzymatic pocket. Barinka et al., JMed Chem; 51(24):7737-7743 (2008). Small halogenated urea compounds suchas DCIBzL and the nearly isosteric DCFPyL, which were designed tointeract with this hydrophobic pocket have very high affinity for PSMA.Chen et al., Clin Cancer Res; 17(24):7645-7653 (2011). Work by Low andco-workers indicated that a thin aminooctanoic acid linker with twophenylalanine residues that interact with a hydrophobic pocket at themouth of the substrate tunnel is correlated with improved binding.Kularatne et al., Mol Pharm; 6(3):790-800 (2009). Zhang et al.discovered an arene binding site near the entrance to the substratetunnel that can be used for constructing high affinity ligands. Zhang etal., J Am Chem Soc; 132(36):12711-12716 (2010). More recently, Eder etal. reported a high affinity PSMA targeting tracer with a lipophilicacyclic Ga(III) chelatorN,N′-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N′-diaceticacid (HBED-CC). Like many groups working on PSMA tracers, the inventorswere focused on binding affinity and the high affinity of RBI-1033 toPSMA was especially attractive. Prior efforts to create a radiolabeledtracer based on RBI-1033 were not successful. The inventors then workedon transforming the 2-5 Å ligand into a peptide equivalent. Theygenerated tri-glutamate (compound 1) and tri-tyrosine (compound 2)mimics of RBI-1033. Neither compound came close to matching the affinityof RBI-1033 which exhibits 10 times better affinity than its parentligand ZJ24. Cramer et al., Nucleosides Nucleotides Nucleic Acids;26(10-12):1471-1477 (2007).

Although the affinity of Compounds 1 and 2 do not match that ofRBI-1033, the fact that one can place multiple negative charges on thelinker without significant reduction in affinity points to a novelengineering pathway for PSMA tracers. Tomachev et al. employed negativecharges in a molecule to reduce liver background of Affibody type ofmolecules. Tolmachev et al., Bioconjug Chem; 21(11):2013-2022 (2010).Affibody molecules are relatively large 7 kD polypeptides where thesurface electrostatics of few amino acids may be manipulated withoutimpacting affinity to targets. The effect of charge placement was lesscertain in a small molecule. The inventors were thus satisfied with theaffinity of Compound 1. They proceeded to attach radiolabels to thenegatively charged framework. The initial biodistribution study with^(99m)Tc labeling and a Gly-Gly-Cys N3S1 chelate indicated significantbackground due to pertechnetate contamination and the need for aradiochemically pure preparation. After redesigning and radiolabelingthe molecule with aluminum-fluoride-NOTA complexes, they were able todemonstrate that this highly negatively charged tracer showssubstantially reduced background compared to published values forexisting tracers. The liver background of [Al¹⁸F]5 is 0.17% ID/g at 2hours compared to 2.17% ID/g for ¹²³I MIP-1072, and 2.1% for ¹⁸F-DCFBCand ¹⁸F-DCFPyL. Chen et al., Clin Cancer Res 17(24): 7645-7653 (2011).Blood pool activity 2 hours post injection is 0.04% ID/g for [Al¹⁸F]5compared to 0.4% ID/g for ¹⁸FDCFBC and ¹⁸FDCFPyL and 0.21% ID/g for ¹²³IMIP-1072. The background in spleen, muscle and bone are very low aswell. In fact, [Al¹⁸F]5 is the only molecule among published PSMAtargeting tracers for PET with consistently low background in all majororgans except kidney and bladder.

Thus, it appears that high affinity was traded for better backgroundclearance. Such a trade-off is possible because of the unique qualitiesof PSMA. PSMA has evolved to catalyze the cleavage of peptidescontaining c-terminal glutamate, so the tunnel is very receptive tonegatively charged molecules. This unique property of PSMA allows one todesign highly negatively charged substrates with adequate affinity.

It is possible that the fast background clearance is due to negativecharges on the NOTA chelate, which contains three carboxylic acidmoieties. Although the acid group on NOTA contributes to the overallhydrophilicity, the chelate itself cannot explain rapid backgroundclearance. In comparison with published tracers with analuminum-fluoride-NOTA complex, [Al¹⁸F]5 has low background as well. Forexample, Lang et. al. reported that alpha-v-beta-3 integrin targeting[¹⁸F]FAI-NOTA-PRGD2 has liver uptake of 1.28±0.28% ID/g at 2 hours andmuscle uptake of 0.68±0.06% ID/g at 2 hours. Lang et al., BioconjugChem; 22(12):2415-2422 (2011). This is well above the liver level of0.17±0.03% ID/g and muscle level of 0.01% ID/g for [Al¹⁸F]5. Lavermanet. al. developed a somatostatin receptor targeting ¹⁸F-IMP466 which hasblood levels of 0.10±0.07% ID/g and a bone level of 0.33±0.07% ID/g at 2hours post injection. Laverman et al., Tumour Biol; 33(2):427-434(2012). These levels are above the blood level of 0.04±0.01% ID/g andbone level of 0.10±0.02% ID/g for [Al¹⁸F]5 at 2 hours post injection.

Tumor uptake vs. background: The tumor uptake amounts of [Al¹⁸F]5 and[^(99m)Tc]3 are comparable to other published ⁶⁸Ga and ^(99m)Tc labeledPSMA tracers. Banerjee et al., J Med Chem; 53(14):5333-5341 (2010). Theuptake is not as high as that of slightly hydrophobic tracers such as¹⁸FDCFPyL nor ¹²³I-MIP-1072. Hillier et al., Cancer Res;69(17):6932-6940 (2009). This could be because ¹⁸FDCFPyL and¹²³I-MIP-1072 have higher affinity to PSMA compared to the highlynegatively charged tracers presented in this paper. Another, possiblymore important, contributing factor could be the fact that most of theinjected tracer molecules are excreted by the kidney before they canmake contact with the tumor. Because the tracer is rapidly excreted,lower tumor uptake resulted, but this also improved backgroundclearance. [Al¹⁸F]5 has the highest tumor to background ratios amongpublished tracers at 2 hours post injection (tumor/blood=115,tumor/liver=27 and tumor/spleen=46, tumor/bone=46 and tumor/muscle=458).This rapid excretion makes the tracer optimal for positron emissiontomography with short-lived radionuclides such as ¹⁸F and ⁶⁸Ga becauseof limited time available for background clearance.

Another possible application for these rapidly excreted PSMA-targetingtracers would be for imaging tumor angiogenesis. PSMA is over-expressedin the lumen of tumor neovasculature. Unlike the PSMA expressed on tumorcells, which is located in the interstitial space, the PSMA in tumorvasculature is within the intravascular space. A tracer injectedintravenously would have direct and rapid access to the intravascularbinding sites. There would be no need for the tracer to diffuse from theintravascular to the interstitial space. The determining factor for thetarget/background ratio would be background clearance. Rapid backgroundclearance was the guiding principle for the development of glycosylatedRGD containing peptides for imaging the alpha-v-beta-3 integrinexpression associated with angiogenesis. Haubner et al., J Nucl Med;42(2):326-336 (2001). ¹⁸F-Galacto-RGD, one of the best studied RGD basedagents, demonstrated background levels of 0.30±0.20% ID/g in blood and2.23±1.02% ID/g in liver at 90 minutes post injection in mice. Pohle etal., Nuclear medicine and biology; 39(6):777-784 (2012).⁶⁸Ga-NODAGA-RGD, a Galacto-RGD derivative with improved eliminationkinetics, has background levels of 0.09±0.03% ID/g in blood and1.58±0.19% ID/g in liver. In comparison, [Al¹⁸F]5 had background levelsof 0.04±0.01% ID/g in blood and 0.17±0.03% ID/g in the liver at 120minutes. The preliminary 1 hour data for [Al¹⁸F]5 also showed lowbackground levels with approximately 0.18% ID/g in the blood and 0.4% inthe liver. [Al¹⁸F]5 may have better elimination kinetics than¹⁸F-Galacto-RGD. Combined with the fact that there is more restrictedexpression of PSMA in tumors and vasculature relative to alpha-v-beta-3,[Al¹⁸F]5 could potentially be a better agent for quantifying tumorangiogenesis.

The study described herein is limited by the number of animals in thebiodistribution study. Two PC3-PIP tumors were used per mouse to getbetter tumor uptake statistics with the limited number of mice. Theinventors intend to perform a more extensive biodistribution study of[Al¹⁸F]5 with more mice and with both PC3-PIP (PSMA+) and PC3-flu(PSMA−) tumors in the same mouse. The inventors focused theirpreliminary biodistribution study on comparing tumor tissues whichexpress PSMA and normal (non-PSMA-expressing) tissues. Having both PIPand flu tumors in the same mouse will allow quantitative comparison ofuptake between the PSMA+ and the PSMA− tumors.

Conclusion:

The inventors have demonstrated that PSMA tracers can be designed with ahighly negatively charged linker. They found that such molecules arerapidly excreted through the kidneys and show minimal nonspecificbinding. The biodistribution properties of [Al¹⁸F]5 suggest that it hasgreat potential for use with positron emission tomography to image PSMAexpressing prostate cancer as well as PSMA expressed in tumorvasculature.

Example 2 Robotic Real-Time Near Infrared Targeted Fluorescence Imagingin a Murine Model of Prostate Cancer

The use of near-infrared (NIR) fluorescence is a promising approach forbiomedical imaging in living tissue. NIR fluorescence (700-1000 nm)detection avoids the natural background fluorescence interference ofbiomolecules, providing a high contrast between target and backgroundtissues. Recently, the Food and Drug Administration (FDA) approved formarketing the Intuitive Surgical da Vinci Fluorescence Imaging VisionSystem, an integration of the SPY imaging technology (NovadaqTechnologies, Mississauga, ON, Canada) into the 3-D high-definitionimaging capabilities of the da Vinci Surgical Robotic System.

Use of the scope in urology has so far been limited to renal surgery, askidney fluorescence is intense after intravenous administration of anontargeted agent such as indocyanine green. Conversely, the prostatedoes not fluoresce after intravenous administration of a nontargetedagent, and therefore the fluorophore must be modified to specificallybind the prostate.

Prostate-specific membrane antigen (PSMA) is a cell surface glycoproteinwith a molecular weight of approximately 100 kDa. It is not expressed insignificant amounts in the prostates of mice, dogs, or monkeys. PSMA isexpressed in high levels in the human prostate, especially in prostatecancer cells and in the vasculature of primary and metastatic prostatetumors.

In this study, the real-time detection of NIR fluorescence emitted by aPSMA-targeted agent was evaluated using the Intuitive da VinciFluorescence Imaging Vision System.

Material and Methods PSMA Ligand

The PSMA targeting ligand ZJ-MCC-dEdEdEGK(IRDye800cw)G is synthesizedusing well-established fluorenylmethyloxycarbonyl (FMOC) solid-phasepeptide synthesis chemistry. Using a rink-amide or equivalent resin,peptide synthesis started with a stem containing theDGlu-DGlu-DGlu-Gly-Lys-Gly sequence. At the N-terminal end of the stem,the linker succinimidyl-4-(Nmaleimidomethyl)cyclohexane-1-carboxylate[SMCC], Thermo Scientific) was added. Then a glutamate and cysteinecontaining urea compound (R)-Cys-C(O)—(S)-Glu was added. Kozikowski etal., J Med Chem.; 44:298-301 (2001). After coupling, the entire compoundwas then cleaved from the solid phase, deprotected with 95%trifluoroacetic acid and purified. An activated ester of IRDye800cw(Li-Cor—BioScience, Lincoln, Nebr.), was then reacted with the primaryamine on the lysine to create the final tracer (FIG. 4). The finalproduct was purified by high-performance liquid chromatography (HPLC)and reconstituted in 0.2 mL of phosphate-buffered saline, pH 7.

Injection of PSMA-Ligand and Preoperative NIR Imaging

PC3-pip (PSMA-positive) and PC3-flu (PSMA-negative) cells were injectedsubcutaneously 1.0×10⁶ PC3 cells with 200 L of matrigel into each flankof 6 NOD SCID Gamma (NSG) mice. Chang et al., Cancer Res.; 59:3192-3198(1999). After the tumors reached a size of ˜5 mm, the mice wereanesthetized with isoflurane and were administered 2 or 10 nmol ofPSMA-binding fluorescent conjugate via tail vein injection. PreoperativeNIR imaging was performed using the Maestro in vivo imaging system(Cambridge Research and Instrumentation, Hopkinton, Mass.). The micewere imaged immediately and at 1-hour intervals after injection of thePSMA targeting fluorescent compound for up to 4 hours. The mice werekilled once PSMA+ and PSMA-tumors could be visually differentiated byNIR imaging. The method used was CO₂ asphyxiation followed by cervicaldislocation.

NIR Fluorescence-Guided Robotic Surgery

Mouse carcasses were operated on using a da Vinci Si Robot (IntuitiveSurgical, Sunnyvale, Calif.). A portable dark box was used to minimizeambient light and to allow optimal fluorescence detection. Three roboticinstruments (the novel endoscope to detect NIR fluorescence, a scissor,and a grasper) were inserted through robotic trocars positioned acrossthe top cover of the dark box. The tumors were excised using the newrobotic camera to detect the fluorescence of the PSMA-binding conjugate.All procedures were done by a single surgeon (R.S.), who had extensiveexperience in robotic surgery. Tumor specimens were sent forhistopathological analysis to assess margin status.

Results

All mice were male, with an average weight of 22 g and a mean age of 49days by the time that they were killed. The mean size of the tumors was5.4 mm. In the first 3 mice, 120 minutes was identified as the time todetect peak fluorescence from the PSMA-positive tumors with the Maestroimaging system (FIG. 5). Within 240 minutes, the signal intensity fromthe tumor was already partially decreased. Based on this information,the last 3 mice were killed at 120 minutes after injection of theconjugate and immediately underwent robotic surgery. Two of the 3 miceinjected with 10 nmol of fluorescent compound demonstrated propertargeting on presurgical NIR imaging, whereas the third mousedemonstrated minimal targeting.

During robotic surgery, the inventors were able to detect fluorescencefrom the PSMA positive tumors in 2 of 3 mice that were injected with 10nmol of fluorescent compound. However, the intensity of fluorescence wasweak (FIG. 6). NIR fluorescence in the base of PSMA positive or PSMAnegative tumors was not observed. In the PSMA negative (control) tumors,no tumor fluorescence was identified in any of the mice. In thesubcutaneous tissue, the fluorescence was limited to the PSMA positivetumors, and a loss of contrast with surrounding tissues was notobserved. Because of the compound's biodistribution and clearance,fluorescence from internal organs (liver, kidneys, and bladder) couldalso be observed, even without penetrating the abdominal wallmusculature, but it did not impair the visualization of the subcutaneoustumor. Given the poor biodistribution of the described fluorescentmarker in tissues not directly targeted, loss of contrast compared withsurrounding tissues is possible, yet unlikely, with higherconcentrations of administered conjugate. Nevertheless, furtherevaluation is needed to test this hypothesis.

PSMA-positive and -negative tumors had the same gross appearance, exceptfor detectable fluorescence in two of the PSMA positive tumors. Nomicroscopic analysis was performed during resection. Microscopy wasobtained later at histopathologic examination, and no fluorescenceimaging was performed at that time. The microscopic appearance ofPSMA-positive and -negative tumors was indistinguishable. Pathologicexamination confirmed prostate cancer and identified negative margins inall 3 PSMA-positive tumors. One of the PSMA-negative tumors had apositive margin.

COMMENT

The inventors were able to detect NIR fluorescence from prostate cancerimplanted in mice with 2 different imaging systems after the intravenousinjection of a new compound targeted to PSMA. The prostate tumors werethen resected using the Da Vinci Si robot equipped with the FluorescenceImaging Vision System, and tumor fluorescence was noted in 2 of 3 mice.This is the first description of fluorescence detection from prostatecancer stained with a PSMA-targeted agent by an imaging system developedfor minimally invasive surgery.

The real-time intraoperative enhanced visualization of organs andtissues with NIR fluorescent dyes has enormous potential clinicalapplications, and one of its most promising uses is in oncology. Few NIRfluorophores, such as indocyanine green (ICG) and methylene blue (MB),are approved by the FDA and available for clinical use. In vivo opticalimaging probes aiming to identify prostatic tissue intraoperatively areunder intense investigation by several groups. PSMA is a potentialtarget for both imaging and treatment purposes. Chen et al synthesizedYC-27, also a PSMA-based imaging agent and conjugated it to IRDye 800CW.Chen et al., Biochem Biophys Res Commun.; 390:624-629 (2009). Eifler etal identified in vitro and in vivo NIR fluorescence imaging of LNCaP andPC3-PIP cells using the Fluobeam (Fluoptics, Grenoble, France), whichcan be used to detect NIR fluorescence during open surgery. Eifler etal., J Urol.; 185:e650-e651 (2011). Liu et al developed Cy5.5-CTT-54.2,another PSMA-targeted NIR fluorescent imaging probe, and identified invitro NIR fluorescence of LNCaP cells. Liu et al. Bioorg Med Chem Lett.;20:7124-7126 (2010). Recently, Nakajima et al synthesized aPSMA-targeted activatable monoclonal antibody fluorophore conjugate(J591-ICG) and detected both in vitro and in vivo fluorescence ofPC3-PIP cells. Nakajima et al., Bioconjug Chem.; 22:1700-1705 (2011).These new imaging probes, as well as the inventors' conjugate, shouldundergo toxicity studies before initiating clinical investigation.

Gordetsky et al investigated the use of NIR fluorescence lymph nodeimaging with open surgery in 14 patients with bladder cancer, using ICGinjected at the tumor base. Gordetsky et al., J Urol.; 185:e308-e309(2011). They used the SPY imaging system (Novadaq Technologies,Mississauga, ON, Canada) to detect fluorescence in pelvic lymph nodespecimens after 2-4 hours. They were able to identify from 1 to 14 lymphnodes per patient in more than 85% of the patients. One lymph node waspositive for high-grade urothelial carcinoma. Van der Poel et alreported the use of a hybrid multimodal radiocolloid(ICG-99mTc-NanoColl) that is both radioactive and fluorescent. van derPoel et al., Eur Urol.; 60:826-833 (2011). After preoperativeintraprostatic injection of the tracer under transrectal ultrasoundguidance and removal of the prostate, they dissected the sentinel lymphnodes guided by a laparoscopic gamma probe (Europrobe, London, UK) and afluorescence laparoscope (Karl Storz, Tuttlingen, Germany), being ableto link preoperative single-photon emission computed tomography/computedtomography (SPECT/CT) guidance with intraoperative NIR fluorescencelaparoscopy.

Several NIR fluorescent dyes have been developed, with properties thatenable them to be conjugated to ligands or monoclonal antibodiesdirected to certain targets, producing agents molecularly specific todetect cancer cells. The increased tumor angiogenesis and expression ofgrowth signaling receptors are key features that can be used to identifyoptimal targets. IRDye 800CW, Cy7, and Alexa Fluor 750 are some of themost commonly used fluorophores. The excitation and emission maxima ofthe IRDye 800CW are centered at 800 nm, which is the optimal wavelengthfor in vivo imaging, with minimal tissue absorption, autofluorescence,and scattering, yielding excellent signal to background ratios (SBR).Kovar et al., Anal Biochem.; 367:1-12 (2007). IRDye 800CW is highlywater soluble and shows very low nonspecific binding to cellularcomponents. Because of their biodistribution and clearance, mostfluorescent agents have a high background signal in the kidneys,bladder, and liver. Keereweer et al., Mol Imaging Biol.; 13:199-207(2011). This was observed in the experiment.

Marshall et al reported no pathologic evidence of toxicity of IRDye800CW based on hematological, biochemistry, and histopathologicalanalyses. Marshall et al., Mol Imaging Biol.; 12:583-594 (2010).However, linking the fluorescent dye and the targeting moiety produces anew molecule that may have properties different from its originalprecursors. Thus, NIR contrast agents should undergo toxicity studies ofthe dye, the targeting ligand, and the final molecule before consideringclinical application.

For open surgery, some of the NIR camera systems developed forimage-guided procedures are the SPY imaging system (NovadaqTechnologies, Mississauga, ON, Canada), the Photodynamic Eye (HamamatsuPhotonics, Hamamatsu City, Japan), the Fluobeam (Fluoptics, Grenoble,France), and the Fluorescence-assisted Resection and Exploration (FLARE)and the Mini-FLARE (Fragioni Laboratory, Brookline, Mass.). In 2011,Intuitive Surgical received FDA approval to market the da VinciFluorescence Imaging Vision System. Tobis et al evaluated the new systemin 11 patients who underwent robotassisted laparoscopic partialnephrectomy (RALP), using intravenous ICG as the NIR fluorophore. Tobiset al., J Urol.; 186:47-52 (2011). Of the 10 malignant tumors, 70% werehypofluorescent and 30% were isofluorescent in comparison with thenormal parenchyma. The vascular anatomy was accurately delineated in allcases with this imaging method.

Fluorescence of organs, such as kidney and liver are straightforward, asnonspecific NIR fluorophores are significantly taken up by these tissuesduring normal clearance. The prostate, by contrast, does not retain asignificant amount of nonspecific fluorescent probe, and thereforeintraoperative fluorescence guidance is not possible. The inventors thussought to develop a targeted fluorophore conjugate that wouldspecifically bind prostatic tissue and lead to detectable levels offluorescence.

The use of implanted tumors derived from prostate cancer cell lines waschosen, as animals do not express significant levels of PSMA in theprostate. The inventors operated on mouse carcasses instead of liveanimals, as they were able to observe NIR fluorescence with the Maestrosystem up to 48 hours after euthanasia in a previous study.

Although NIR fluorescence from prostate tumors was detected with boththe Maestro in vivo imaging system and the new robotic scope, thefluorescent signal was considerably less with the robotic system. Thefluorescence of the tumor noted by the robotic system was diffuse butwith some patchy distribution. Because of the lack of more intensefluorescent staining, it is difficult to completely characterize thestaining pattern of individual tissue components. With more intensestaining, a complete assessment of the staining pattern should beperformed. Several factors could possibly explain the decreasedfluorescent signal noted with the robotic system. The camera of theMaestro system can be selected to receive longer periods of exposure toNIR fluorescence, which permits stronger signal detection but is notideal for real-time image guidance. By contrast, the frame rate of therobotic scope is 30 frames per second, which was adequate for real-timedetection of NIR fluorescence from other organs such as the kidney, butmay not be optimal to detect fluorescence from prostate tissue with theinventors' tracer. Another possible explanation is that theconcentration of 10 nmol, used in the experiments, may not be enough forstrong real-time NIR fluorescence detection in prostatic tissue. Onealso cannot exclude the possibility that a certain amount of the tracerdid not reach the intravascular space during the tail vein injection,which could have led to less accumulation in the tumor, impairing itsvisualization. Another limitation is the small sample size. However,this was mainly a proof-of-concept study, as it was the first time thatthe robotic scope was used to detect fluorescence from the PSMA-targetedconjugate.

Even without detecting fluorescence intensity as strong as that seen inthe kidneys and bladder, the preliminary results are encouraging.Further studies will be useful to more precisely define the role of NIRfluorescence image-guided robotic surgery, identifying which procedureswill be the most suitable for its application. The cause for thepositive margins in 1 tumor cannot be precisely determined. Althoughevidence from this study is not intended or adequate to draw conclusionsregarding the improvement of margin status, with further refinement offluorescent visualization, the precision of tumor resection couldpotentially increase, minimizing damage to normal tissue and ideallyimproving tumor margin assessment intraoperatively. Intraoperativerobotic fluorescence detection in real time with nonspecificfluorophores, such as ICG, is readily available for clinical use. In thefuture, other fluorescent dyes suitable for binding to ligands that canbe targeted to specific molecules may also obtain approval for clinicaluse, broadening the spectrum of procedures which can be enhanced by NIRfluorescence-guided surgery.

CONCLUSIONS

This example demonstrates the feasibility of prostatic tissueidentification and resection by using a novel robotic fluorescenceimaging system in a murine model after the intravenous injection of aPSMA-targeted agent. Further research is warranted to improve real-timefluorescence detection of prostatic tissue and to move this technologytoward its potential clinical application.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material cited herein areincorporated by reference. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. The invention isnot limited to the exact details shown and described, for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

What is claimed is:
 1. A PSMA-specific imaging agent comprising acompound according to formula I:

wherein S¹ is an organic spacer group having from 5 to 30 carbons, A isan amino acid forming a portion of a negatively charged peptideoligomer, n is from 3 to 6, S² is an organic spacer group having from 5to 15 carbons, and I is an imaging group, and pharmaceuticallyacceptable salts thereof.
 2. The imaging agent of claim 1, wherein theamino acid is glutamic acid.
 3. The imaging agent of claim 1, wherein nis
 3. 4. The imaging agent of claim 1, wherein the organic spacerscomprise one or more amino acids.
 5. The imaging agent of claim 1,wherein S¹ is an organic spacer group having from 10 to 15 carbons. 6.The imaging agent of claim 1, wherein S¹ is an organic spacer grouphaving the structure:


7. The imaging agent of claim 1, wherein the imaging group is anear-infrared imaging group.
 8. The imaging agent of claim 7, whereinthe imaging agent is ZJ-MCC-dEdEdEGK(IRDye800cw)G.
 9. The imaging agentof claim 1, wherein the imaging group is a positron or single-photonemission tomography imaging group.
 10. The imaging agent of claim 9,wherein the imaging agent is selected from the group consisting ofE′E-Amc-Ahx-dEdEdEGYGGGC-NH₂, E′E-Amc-Ahx-dEdEdEYK(Bn-NOTA)-NH₂,E′E-Ahx-EEEYK(Bn-NOTA)-NH₂.
 11. A method for imaging prostate cancer ina tissue region of a subject comprising: (a) administering to thesubject a detectably effective amount of a PSMA-specific imaging agentcomprising a compound according to formula I:

wherein S¹ is an organic spacer group having from 5 to 30 carbons, A isan amino acid forming a portion of a negatively charged peptideoligomer, n is from 3 to 6, S² is an organic spacer group having from 5to 15 carbons, and I is an imaging group, and pharmaceuticallyacceptable salts thereof; (b) allowing a sufficient amount of time forthe PSMA-specific imaging agent to enter the tissue region; and (c)performing imaging of the tissue region of the subject using an imagingdevice capable of detecting the imaging group.
 12. The method of claim11, wherein the tissue region is the prostate gland.
 13. The method ofclaim 11, wherein the amino acid of the imaging agent is glutamic acid.14. The method of claim 11, wherein n of the imaging agent is
 3. 15. Themethod of claim 11, wherein S¹ of the imaging agent is an organic spacergroup having from 10 to 15 carbons.
 16. The method of claim 11, whereinthe imaging device is a positron or single-photon emissiontomography/computed tomography scanner, and the imaging group is acorresponding positron or single-photon emission tomography imaginggroup.
 17. The method of claim 16, wherein the imaging agent is selectedfrom the group consisting of E′E-Amc-Ahx-dEdEdEGYGGGC-NH₂,E′E-Amc-Ahx-dEdEdEYK(Bn-NOTA)-NH₂, E′E-Ahx-EEEYK(Bn-NOTA)-NH₂.
 18. Themethod of claim 11, wherein the imaging device is near-infrared imagingdevice, and the imaging group of the imaging agent is a near-infraredimaging group.
 19. The method of claim 18, wherein the imaging agent isZJ-MCC-dEdEdEGK(IRDye800cw)G.
 20. The method of claim 11, furthercomprising the step of conducting surgery to remove prostate cancertissue from the subject.
 21. The method of claim 20, wherein the surgeryis near-infrared fluorescence-guided robotic surgery.